The highly functionalized diterpenoid Taxol (Wani et al., J. Am. Chem. Soc. 93:2325-2327, 1971) is well-established as a potent chemotherapeutic agent (Holmes et al., in Taxane Anticancer Agents: Basic Science and Current Status, Georg et al., eds., pp. 31-57, American Chemical Society, Washington, D.C., 1995; Arbuck and Blaylock, in Taxol: Science and Applications, Suffness, ed., pp. 379-415, CRC Press, Boca Raton, Fla., 1995). (Paclitaxel is the generic name for Taxol, a registered trademark of Bristol-Myers Squibb.)
The supply of Taxol from the original source, the bark of the Pacific yew (Taxus brevifolia Nutt.; Taxaceae) is limited. As a result, there have been intensive efforts to develop alternate means of production, including isolation from the foliage and other renewable tissues of plantation-grown Taxus species, biosynthesis in tissue culture systems, and semisynthesis of Taxol and its analogs from advanced taxane diterpenoid (taxoid) metabolites that are more readily available (Cragg et al., J. Nat. Prod. 56:1657-1668, 1993). Total synthesis of Taxol, at present, is not commercially viable (Borman, Chem. Eng. News 72(7):32-34, 1994), and it is clear that in the foreseeable future the supply of Taxol and its synthetically useful progenitors must rely on biological methods of production, either in Taxus plants or in cell cultures derived therefrom (Suffness, in Taxane Anticancer Agents: Basic Science and Current Status, Georg et al., eds., American Chemical Society, Washington, D.C., 1995, pp. 1-17).
The biosynthesis of Taxol involves the initial cyclization of geranylgeranyl diphosphate, the universal precursor of diterpenoids (West, in Biosynthesis of Isoprenoid Compounds, Porter and Spurgeon, eds., vol. 1, pp. 375-411, Wiley & Sons, New York, N.Y., 1981), to taxa-4(5),11(12)-diene (Koepp et al., J. Biol. Chem. 270:8686-8690, 1995) followed by extensive oxidative modification of this olefin (Koepp et al., J. Biol. Chem. 270:8686-8690, 1995; Croteau et al., in Taxane Anticancer Agents: Basic Science and Current Status, Georg et al., eds., pp. 72-80, American Chemical Society, Washington, D.C., 1995) and elaboration of the side chains (FIG. 1) (Floss and Mocek, in Taxol: Science and Applications, Suffness, ed., pp. 191-208, CRC Press, Boca Raton, Fla., 1995).
Taxa-4(5),11(12)-diene synthase ("taxadiene synthase"), the enzyme responsible for the initial cyclization of geranylgeranyl diphosphate, to delineate the taxane skeleton, has been isolated from T. brevifolia stem tissue, partially purified, and characterized (Hezari et al., Arch. Biochem. Biophys. 322:437-444, 1995).
Although taxadiene synthase resembles other plant terpenoid cyclases in general enzymatic properties (Hezari et al., Arch. Biochem. Biophys. 322:437-444, 1995), it has proved extremely difficult to purify in sufficient amounts for antibody preparation or microsequencing, thwarting this approach toward cDNA cloning.